Rankl-specific agent for treating metastatic disease

ABSTRACT

The invention provides for a RANKL-specific antagonistic agent recognizing human platelet-expressed receptor activator of nuclear factor kappa-B ligand (pRANKL), for use in treating a cancer patient to prevent or reduce premetastatic lesions in blood.

TECHNICAL FIELD

The invention refers to a RANKL-specific antagonistic agent whichrecognizes and optionally neutralizes human platelet-expressed receptoractivator of nuclear factor kappa-B ligand (pRANKL), for use in treatinga cancer patient, a method for identifying a lead candidate agent, and amethod of predicting the metastatic potential in a cancer patient.

BACKGROUND

A large field of research and development focuses on the treatment ofcancer. Products under development range from kinase inhibitors, toangiogenesis inhibitors, monoclonal antibodies against tumor targets,apoptosis inducers, anti-tumor vaccination, and conventionalchemotherapeutic agents against various tumor targets and with variouscytotoxic effects. Prognosis of cancer patients is mainly determined bythe risk of developing metastasis.

During metastasis, host cells are recruited to disseminated tumor cellsto form specialized microenvironments (“niches”) that promote metastaticprogression, but the mechanisms guiding the assembly of these niches arelargely unknown. Labelle et al. (PNAS 2014, E3053-3061) describe e.g.that platelet-derived rather than tumor cell-derived signals arerequired for the rapid recruitment of granulocytes to tumor cells toform “early metastatic niches.” Platelets are described to interact withtumor cells during their transit through the circulation thereby formingplatelet-tumor cell aggregates and would enhance metastasis via multiplemechanism.

Gay et al. (Nature Reviews 2011, 11:123-134) review the contribution ofplatelets to tumor metastasis. Among others, within the circulatorysystem, platelets would guard tumor cells from immune elimination andpromote their arrest at the endothelium, supporting the establishment ofsecondary lesions. The adhesion of platelets to tumor cells and theirincorporation into platelet heteroaggregates is described to shield thetumor cells from NK cell activity.

Bone metastases are a frequent complication of many cancers that resultin severe disease burden and pain. Regulation of cancer cell migrationand bone metastasis by RANK (receptor activator of NF-kB) ligand (RANKL)is described by Jones et al. (Nature 2006, 440:692-696). RANKL triggersmigration of human epithelial cancer cells and melanoma cells thatexpress the receptor RANK. RANK is expressed on a series of cancer celllines and cancer cells in patients. In a mouse model of melanomametastasis, in vivo neutralization of RANKL by osteoprotegerin resultsin complete protection from paralysis and a marked reduction in tumorburden in bones, but not in other organs. RANKL produced by bonemicroenvironment is considered a fertile soil for RANK-positive tumorcells.

Dougall et al. (BoneKEy Reports 2014, 3:519) describes RANKL anessential mediator of osteoclast function and survival, acting throughits cognate receptor, RANK. Preclinical data have firmly establishedthat blockade of tumor-induced osteoclastogenesis by RANKL inhibitionwould not only protect against bone destruction, but would also inhibitthe progression of established bone metastases and delay the formationof de novo bone metastases in cancer models. In patients with bonemetastases, skeletal complications are driven by increased osteoclasticactivity and may result in pathological fractures, spinal cordcompression and the need for radiotherapy to the bone or orthopedicsurgery (collectively known as skeletal-related events (SREs)).Denosumab, a fully human monoclonal antibody against RANKL, is describedto prevent or delay SREs in patients with solid tumors that havemetastasized to bone. In addition to its central role in tumor-inducedosteolysis, bone destruction and skeletal tumor progression, there isemerging evidence for direct prometastatic effects of RANKL, independentof osteoclasts. For example, RANKL also stimulates metastasis viaactivity on RANK-expressing cancer cells, resulting in increasedinvasion and migration.

Tan et al. (Nature 2011, 470 (7335):548-553) describe thatfibroblast-recruited, tumor infiltrating CD4+ T cells stimulate mammarycancer metastasis through RANKL-RANK signalling.

Denosumab was approved by the U.S. Food and Drug Administration for usein postmenopausal women with risk of osteoporosis under the trade nameProlia, and as Xgeva, for the prevention of SREs in patients with bonemetastases from solid tumors. Clinical trials were investigatingDenosumab in giant cell tumors, multiple myeloma with bone metastases,and hypercalcemia of malignancy.

Therapies targeting RANK/RANKL e.g. involve RANKL-specific binders,among them Denosumab, recombinant RANK-Fc (Schmiedel et al. 2013, CancerRes. 73(2):683-94), or RANKL-nanobodies (WO2008142164A2). RANKL-bindingpeptides are described to inhibit bone resorption and/or osteoclastactivity (WO2012163887A1).

The effect of Denosumab on bone metastasis in patients with advancedsolid tumors is described in a series of documents e.g., Rolfo Christianet al. (Expert Opinion on Biological Therapy, vol. 14, no. 1, 2014, pp15-26), Scagliotti Giorgio Vittorio et al. (Journal of ThoracicOncology, vol. 7, no. 12, 2012, pp 1823-1829), Morikawa K. et al.(Database Embase, Elsevier Science publishers, Amsterdam, XP002736136;and Japanese Journal of Lung Cancer, vol. 52, no. 7, 2012, pp1035-1040), Takeshi Yuasa et al. (Oncotargets and Therapy, vol. 5, 2012,pp 221-229), Hilbe Wolfgang et al. (Magazine of European MedicalOncology, AT, vol. 6, no. 2, 2013, pp 75-82), Laszlo Kopper (Pathology &Oncology Research, vol. 18, no. 4, 2012, pp 743-747), Sonya J. Snedecoret al., (Clinical Therapeutics, vol. 34, no. 6, 2012, pp 1334-1349),Sarah Payton (Nature Reviews Urology, vol. 9, no. 1, 2011, pp 1-1), WO2013/176469 and DATABASE WPI, Thomson Scientific, London, GB,XP002736138; US 2012/114665 A1, and WO 01/08699 A1).

Nakanishi et al. (Platelets 2014, Early Online 1-7) describe the role ofplatelets to enhance the Th2 response mediated by dendritic cells (DCs)thereby contributing to allergic inflammation. Thrombin receptor agonistpeptide (TRAP)-activated platelets were found to express RANKL andinduced maturation of myeloid DCs.

B. A. Kerr et al. (Oncogene, vol. 32, no. 36, 2013, pp 4319-4324)describe the role of platelets governing pre-metastatic tumorcommunication to bone.

Sharma Deva et al. (Journal of Cellular Physiology, vol. 229, no. 8,2014, pp 1005-1015) describe the role of platelets in tumor progression.Platelets are described to shield tumor cells from immune host responsesand promote tumor cell survival.

Esposito Mark et al. (Pharmacology and Therapeutics, GB, vol. 151, no.2, pp 222-233) describe targeting tumor stromal interactions in bonemetastasis. It is described that circulating tumor cell survival isenhanced by platelet secretion of TGFbeta and formation of plateletaggregates.

SUMMARY OF THE INVENTION

It is the object of the invention to provide for an improved treatmentof metastatic disease, and respective anti-metastatic agents.

The object is solved by the subject matter of the invention.

The present invention provides for a RANKL-specific antagonistic agentrecognizing human platelet-expressed receptor activator of nuclearfactor kappa-B ligand (pRANKL), for use in treating a cancer patient toprevent or reduce premetastatic leasons in blood. Specifically, themedical use comprises the prevention or reduction of premetastaticcirculating cell aggregates of platelets with cancer cells. Suchaggregates specifically comprise the platelets as activated plateletsexpressing the pRANKL. Specifically, the RANKL-specific antagonisticagent is used in an effective amount to prevent transduction of signalsupon RANK-RANKL interaction that facilitate metastasis upon formation ofsuch premetastatic circulating cell aggregates in blood, or to preventformation of such premetastatic circulating cell aggregates in blood.

In particular, binding and neutralizing pRANKL would inhibit

a) the dissemination of premetastatic tumor cells,

b) the activation of platelets and/or cancer (or tumor) cells to expresspRANKL or to induce RANK-RANKL signaling, thereby inhibiting thetransformation of the cells to become prometastatic, and/or

c) the risk of developing haematogeneous spread optionally followed bymetastasis formation.

pRANKL specifically turns out to be upregulated when interacting withcancer cells. Therefore, inhibition or neutralizing pRANKL by the agentas described herein, would downmodulate the premetastatic lesion.

The premetastatic lesions are typically involving cells of a precursorlesion, which is characterized by changes in the appearance or nature ofthe cell before it becomes cancerous, or in the case of a cancer cellbefore it becomes metastatic.

Therefore, the invention provides for a new method of treatment, whereina cancer patient is treated with the agent in an effective amount toprevent or reduce premetastatic leasons.

Specifically, the cancer cells originate from RANK-positive cancercells. The cancer cells can be RANK-positive tumor-forming cancer cellsor tumor cells, in particular solid tumor cells, or RANK positive cancercells which involve the blood and blood-forming organs, e.g. leukemia.Specifically, premetastatic lesions are identified by determiningactivated platelet-cancer cell aggregates, which are consideredcirculating premetastatic cell clusters forming niches, therebypromoting cancer metastasis or increasing the risk of developingmetastasis, e.g. in distant organs or bone metastasis. Upon interactingwith the activated platelets, the cancer cells may be transformed intoRANKL positive cancer cells, which are a further characteristic of thepremetastatic lesions. Since such platelet-cancer cell aggregates areblood-borne, the metastatic risk or potential is also referred to asblood-borne or haematogeneous.

The agent may specifically recognize and neutralize pRANKL only, orcross-specifically recognizes various forms of RANKL. Specifically, theagent is cross-reactive, recognizing and optionally neutralizing pRANKLand at least one of soluble receptor activator of nuclear factor kappa-Bligand (sRANKL) and membrane-bound activator of nuclear factor kappa-Bligand (mRANKL), or both.

Specifically, the agent is recognizing the RANKL polypeptide, which maycomprise the full amino acid sequence of human RANKL (SEQ ID 3), or anepitope in the extracellular portion of the pRANKL, e.g. AA 69-AA 317 ofSEQ ID 3), in particular competing with the binding of RANK to RANKL orpRANKL and optionally any other form of RANKL, and thereby substantiallyinhibiting the RANK-RANKL signalling.

Specifically, the agent binds to pRANKL, thereby inhibiting pRANKL fromactivating its receptor on cancer cells, e.g. on disseminating ormetastasizing tumor cells.

Specifically, the agent is binding to pRANKL monomer, or multimer, suchas a multimer of RANKL molecules interacting on the surface ofplatelets, e.g. wherein one or more pRANKL molecules are bound on thesurface of the platelets interacting with each other, and/or interactingwith one or more RANKL molecules which are not-platelet bound, orsRANKL. Such multimer may be a dimer, or trimer, or higher multimer,preferably forming a complex with platelet surface-bound pRANKL and/orpRANKL cleaved from the platelet surface, and/or sRANKL, and/or mRANKL.Thus, binding may occur, e.g. on the surface of the platelet, in themicroenvironment between a platelet and a cancer cell, or in thecirculation upon cleavage of the pRANKL from the platelet surface.

Specifically, metastasis is blood borne, with tumor cell aggregation indistant organs, e.g. any of lung, liver, intestine, skin, muscle,spleen, pancreas, kidney, bone, or brain. Specifically, the risk ofdeveloping haematogenic metastatic disease in a patient suffering from aprimary solid tumor or cancer of the blood and the lymphatic system, canbe effectively reduced.

Specifically, the cancer patient is at risk of or suffering from minimalresidual disease and/or recurrence of metastatic disease, optionallywherein the patient has a detectable level of circulating tumor cells ina blood sample, e.g. as determined by the number of disseminated tumorcells in whole blood or a blood fraction thereof, or by specific tumorcell marker. A detectable number of tumor cells is e.g. less than 10, orless than 5, or 4, 3, 2, or 1 circulating tumor cells in a sample ofwhole blood of at least 5 mL, or 7.5 mL, or 10 mL.

According to a specific embodiment, the patient suffers from a solidtumor selected from the group consisting of epithelial tumors andmesenchymal tumors, or tumors of endodermal, mesodermal and/orectodermal origin, or a blood-borne cancer, such as leukemia.

Specifically, the patient suffers from breast cancer, pancreatic cancer,gastric cancer, esophageal cancer, renal cell carcinoma, lung carcinoma,colon/rectal/colorectal cancer, melanoma, prostate cancer, head and neckcancer, or leukemia.

According to a specific aspect, the treatment is combined with surgicalintervention to remove at least part of a tumor, and/or combined withradiotherapy, and the agent is administered for neoadjuvant or adjuvanttherapy. Accordingly, the patient specifically is preparing for orundergoing surgical intervention and/or radiotherapy, or has beentreated by a surgical intervention and/or radiotherapy, and is furthertreated with the agent according to the invention before or aftersurgery. According to specific examples, such treatment may startbetween 1 to 30 days before surgery, or during surgery, or within 1 to30 days after surgery, and the agent may be administered for a continuedperiod, e.g. for 1 to 12 months, or even longer, wherein the agent isadministered in regular intervals. Surgical interventions are e.g.therapeutic removal of tumor mass, or biopsy. Surgery is considered aspecific risk factor of disseminating tumor cells into the blood stream,thereby provoking platelet-tumor cell aggregate formation. Likewise,radiation therapy can trigger the haematogeneous spread of tumor cells.Therefore, the method of the invention specifically is indicated incombination with surgery and/or radiotherapy which potentiallydisseminates solid tumor cells.

According to a specific aspect, the agent is administered to the patientin combination with an adjuvant or neoadjuvant combination therapy,preferably chemotherapy, kinase inhibitor therapy and/or immunotherapy.Such combination therapy would specifically target the cancer cell, e.g.any tumor associated antigen, such as selected from the group consistingof epithelial cancer cell marker, soluble factors, or anti-angiogenictherapy.

According to a specific aspect, the agent is selected from the groupconsisting of antibodies, antibody fragments, receptor-fusion proteins,such as RANK-Fc fusion proteins, peptides, such as inactivated forms ofosteoprotegerin, or fragments thereof, small molecules, such asRANK-specific organic small molecules, or aptamers. Exemplary smallmolecules are small molecule inhibitors of RANKL and TNF, such asdescribed in Coste E, et al. Ann Rheum Dis 2015; 74:220-226.doi:10.1136/annrheumdis-2013-203700. Specific examples are derivativesof butanediol biphenylcarboxylic acid ester, which are capable ofinhibiting RANKL-induced phosphorylation of IκB and extracellularsignal-regulated kinase (ERK). For example, compounds where the esterbond is replaced by a ketone may be used, such as ABD328 and ABD345characterized by the following formula:

Specifically, the agent is a human or humanized antibody, such asDenosumab, or a functional variant thereof, or an antigen-bindingfragment of any of the foregoing, or a RANK-Fc fusion protein. Denosumab(Amgen, Thousand Oaks, Calif., USA) is a fully human IgG2 monoclonalantibody specific to RANKL, which is described to suppress boneresorption markers in patients with a variety of metastatic tumors andis being investigated in multiple clinical trials for the prevention andtreatment of bone metastases. Chemically, it consists of 2 heavy and 2light chains. Each light chain consists of 215 amino acids. Each heavychain consists of 448 amino acids with 4 intramolecular disulfides. Theheavy chain amino acid sequence is identified by SEQ ID 1; the lightchain amino acid sequence is identified by SEQ ID 2.

Specifically, the agent comprises an Fc antibody fragment, such as ahuman IgG1 Fc, which is engineered to reduce Fc effector function (e.g.which does not significantly bind to the FcgammaRIIIa, or CD16), andtherefore does not exhibit significant antibody-dependent cellularcytotoxicity (ADCC). Exemplary Fc fragments which comprise pointmutations to reduce Fc effector function are characterized by at leastone of the following mutations: E233P, L234V, L235A, deltaG236, A327G,A330S, wherein nomenclature is according to the EU index of Kabat.

Alternatively, the agent comprises an Fc antibody fragment, such as ahuman IgG1 Fc, with Fc effector function (e.g. binding to theFcgammaRIIIa, or CD16), such as ADCC. Such agent would have theadditional advantage of cell-mediated immune defense whereby an effectorcell of the immune system actively destroys the target cell, which isthe platelet and/or the cancer cell, preferably the cancer-plateletaggregate.

According to a specific aspect, the agent is administered to the patientin a therapeutically effective amount by systemic administration,preferably by intravenous infusion or bolus injection.

Prior art therapy with Denosumab would typically involve subcutaneoustreatment. The present invention would target activated circulatingplatelets expressing RANKL, or circulating platelet-cancer cellaggregates. Therefore, the intravenous route is specifically preferred.

Preferred doses are, e.g. ranging from 0.5 to 1000 mg, preferably 1-400mg. If administered subcutaneously, the preferred dosage is ranging from0.5 to 400 mg.

The invention further provides for a method for identifying a leadcandidate agent that is effective in preventing or treatingpremetastatic lesions, or minimal residual disease and/or recurrence ofmetastatic disease in a cancer patient, such as a patient preparing foror undergoing surgical intervention to remove at least part of a solidtumor and/or radiotherapy, or who has undergone such surgicalintervention and/or radiotherapy, the method comprising screening one ormore test agents in a cell-based assay, which assay comprises the steps:

a) providing a cancer cell culture;

b) contacting the cell culture with human blood platelets in a reactionmixture with a test agent; and

c) detecting if the test agent

-   -   i) inhibits RANK signalling by pRANKL; and/or    -   ii) decreases the level of platelet binding to the cancer cells;

thereby identifying a lead candidate agent for preventing or treatingpremetastatic lesions, and optionally its potential to prevent or treatminimal residual disease and/or recurrence of metastatic disease in acancer patient.

Specifically, the detection step c) involves testing of both, if thetest agent

-   -   i) inhibits RANK signalling by pRANKL; and    -   ii) decreases the level of platelet binding to the cancer cells.

Specifically, activated platelets are used in such screening assay, suchas thrombin-activated platelets, or those activated by cancer or tumorcells, e.g. activated by RANKL-negative tumor cells, to express pRANKL.

Specifically, the assay is a functional antagonist or neutralizing assaymeasuring the ability of a putative antagonist (a test agent) to inhibitreceptor (RANK) signalling mediated by an agonist (RANKL). For example,a compound can be identified as a RANKL-specific antagonistic agent ifthe compound substantially inhibits the receptor-mediated signalling.

The invention further provides for a method of predicting the metastaticpotential in a cancer patient, comprising

a) providing a sample of peripheral blood or a platelet containing bloodfraction;

b) determining the pRANKL expression in said sample and comparing to areference value, the differential expression being indicative ofpremetastatic lesions and an increased potential of developing distantmetastases.

For example, the sample or the platelets can be incubated with standardcancer cells with defined metastatic potential, and the level of RANKsignalling by the platelet-cancer cell interaction may be determined toobtain a reference value for a specific metastatic potential. Likewise,the platelets can be activated with thrombin and the pRANKL level in thesample may be determined in comparison with a standard.

The pRANKL expression is e.g. determined as the level of pRANKLexpression, such as the expression of a nucleotide sequence or thepRANKL polypeptide, or a fragment thereof. The level may be determinedqualitatively, but also semi-quantitatively, or quantitatively.

The reference value may be derived from a positive or negative control,or both. The positive control is e.g. representing the level of pRANKLexpression of platelets from a cancer patient suffering from metastaticdisease conditions. The negative control is e.g. representing the pRANKLexpression level of a healthy control subject.

FIGURES

FIG. 1: Lung metastasis model using RANKL transfected melanoma cells.Murine B16-F10 melanoma cells (ATCC), to be identified by black color)that were transfected to express human RANKL (RANKL+) or control cells(parental) were injected in the tail vein of C57BL/6 mice. B16-F10 cells(100,000 cells per mouse). After passing the heart via the blood stream,the malignant cells disseminate to the lungs. The metastatic burden inthe lung of the animals was analyzed after 3 weeks, showing drasticallyincreased metastasis upon enhanced RANKL-mediated signalling when RANKL+cells were used. This is revealed by the higher amount of black colouredareals in the lungs and the destroyed lung architecture.

FIG. 2: RANKL expression on resting and activated thrombocytes. Humanplatelets of healthy donors were isolated by centrifugation of bloodsamples and then either stimulated with 0.2 U/ml of the classicalplatelet agonist Thrombin (activated) or left untreated (resting).Platelet surface expression of the activation marker P selectin (CD62P)and RANKL was analyzed by flow cytometry.

FIG. 3: Structure of RANK-Fc-KO. RANK-Fc-KO fusion proteins consist ofthe extracellular domain of the human receptor RANK (Q25-P207; Gen BankReference NP_0003830) and a human IgG1 Fc part (P217-K447) containingamino acid exchanges E233P/L234V/L235A/ΔG236/A327G/A330S; Armour et al.2003, Mol Immunol. 40(9):585-93; Schmiedel et al. 2013) to decrease itsaffinity and consequently binding to the Fc receptor CD16. Here, allnumbering is according to Kabat [EU-Index].

FIG. 4: Lung metastasis model using RANK-Fc-KO. Parental B16-F10melanoma cells were injected in the tail vein of the indicated numbersof C57BL/6 mice (75,000 per mouse). Additionally, mice in the differentgroups were treated either with platelet depletion by application of 3μg/g anti-GPIbα antibody 24 h prior to tumor cell injection, RANK-Fc-KO(100 μg per mouse, on the day of tumor cell injection, repeated two andfour days later) as well as appropriate controls (ctrl) as indicated.The number of lung metastasis was counted after sacrifice of mice after3 weeks.

FIG. 5: Prevention of platelet-induced (prometastatic) EMT signaling inimmortalized MCF10A cells by RANKL blocking. MCF10A cells (ATCC) asstandard model for EMT analysis were studied by quantitative realtimePCR for (A) RANKL and (B) the markers for mesenchymal phenotype ZEB(Zink finger E-box binding homeobox 1, left column), and NCadherin(right column). To this end, RNA was isolated, reverse transcribed andsubjected to SYBR-green based PCR by routine techniques.

(A) The ratio of target (RANKL) to reference (RPL13) gene expression ofMCF10A cells and appropriate controls is displayed. The results show nodifference between negative control and MCF10A cells thereby excludingthat MCF10A cells themselves express RANKL.

(B) Analysis of prometastatic EMT gene signatures in MCF10A cells wasperformed after 2 days of culture alone (untreated) or with platelets(ratio platelets/tumor cells 5:1) in the presence or absence of theRANKL-neutralizing antibody Denosumab (10 μg/ml). Presence of plateletscaused induction of ZEB and NCadherin mRNA expression as markers forprometastatic EMT gene expression that was substantially reduced by thepresence of Denosumab.

FIG. 6: Sequences

Denosumab heavy and light chain amino acid sequences:

SEQ ID 1: heavy chain

SEQ ID 2: light chain

Human RANKL amino acid sequence (GenBank: AAB86811.1):

SEQ ID 3: full-length sequence

FIG. 7: Neutralisation of RANKL prevents platelet-induced migration ofimmortalized MCF10A cells. 1*10⁵ MCF10A cells were seeded in the topchamber of a transwell insert (8 μm pore size) either alone (untreated)or with of human platelets (1.5*10⁵/μl) in the presence or absence ofthe RANKL-neutralizing antibody Denosumab or the respective isotypecontrol (each 5 μg/ml). After 24 h of incubation, EGF (20 ng/ml) wasadded to the lower chamber to act as chemoattractant. After a total of48 h incubation, non-motile cells on the upper side of the membrane wereremoved while the migrated cells on the lower side were fixed, stainedwith DAPI and counted under the microscope.

FIG. 8: Lung metastasis model using platelet-specific RANKL knockoutmice. B6.129-Tnfsf11^(tm1.1Caob)/J mice in which the RANKL gene isflanked by loxP sites (hereinafter referred to as RANKL fl/fl) andC57BL/6-Tg(Pf4-cre)Q3Rsko/J mice which contain a megakaryocyte/plateletspecific recombinase (hereinafter referred to as Pf4cre) were obtainedboth from The Jackson Laboratory (Bar Harbor, Me. USA) were bred togenerate RANKL fl/fl Pf4 cre/+knockout (ko) mice in which RANKL isspecifically knocked out in megakaryocytes/platelets. For determinationof the effects of platelet-expressed RANKL, B16-F10 melanoma cells(75,000 per mouse) were injected via the tail vein in RANKL fl/fl Pf4cre/+knockout (ko) mice or C57BL/6 control mice (ctrl) (ko, n=8 animals;ctrl, n=5 animals). The number of lung metastases was counted aftersacrifice of mice after 3 weeks.

FIG. 9: Neutralisation of RANKL prevents platelet-induced prometastaticEMT signaling in SK-Mel melanoma cells. (A) SK-Mel (ATCC) cells wereanalyzed by flow cytometry using anti-RANKL antibody (filled histogram)or the respective isotype control (dotted line) followed byanti-mouse-PE. No difference between isotype control and RANKL-specificantibody-binding was observed, thereby excluding that SK-Mel cellsthemselves express RANKL.

(B) Analysis of prometastatic EMT gene signatures in SK-Mel melanomacells was performed after 1 day of culture alone (untreated) or withplatelets (ratio platelets/tumor cells 200:1) or platelets and theRANKL-neutralizing antibody Denosumab (5 μg/ml). Then the markers formesenchymal phenotype, ZEB (Zink finger E-box binding homeobox 1), Twistand Vimentin were analyzed by quantitative realtime PCR. To this end,RNA was isolated, reverse transcribed and subjected to SYBR-green basedPCR by routine techniques.

Presence of platelets induced prometastatic mRNA expression of all threemarker genes that was substantially reduced by the presence ofDenosumab.

DETAILED DESCRIPTION OF THE INVENTION

The term “adjuvant” as used herein shall refer to the treatment ofcancer during or after a surgical intervention and/or radiotherapy, e.g.for improved therapy.

The term “neoadjuvant” as used herein shall refer to the treatment ofcancer prior to a surgical intervention and/or radiotherapy, e.g. forimproved therapy.

The term “RANKL-specific antagonistic agent” as used herein shall referto a compound, which is a RANKL binder substantially neutralizing RANKL,and/or reducing, or inhibiting binding of RANKL to its receptor RANK,thereby antagonizing the RANK-RANKL signalling pathway.

The antagonistic function of the agent is specifically characterized bydiminishing, inhibiting, or preventing a cellular response to a receptor(RANK) activated by an agonist (RANKL). Antagonists specifically arecompetitive antagonists, which can reversibly bind to the RANKL at thesame binding site or interfering with the binding site (active site), asthe endogenous receptor, without necessarily activating the receptor.

The agent can be any suitable binder or ligand, e.g. selected from thegroup consisting of small organic or inorganic molecules, carbohydrates,biological macromolecules, peptides, proteins (herein also referred toas polypeptides), peptide analogs, peptidomimetics, antibodies,including antigen-binding fragments of antibodies, nucleic acids,nucleic acid analogs, and a combination of any of the foregoing. In someembodiments, the RANKL-specific antagonistic agent is animmunotherapeutic agent. A specific example of the agent is selectedfrom the group consisting of an antibody, a receptor or osteoprotegerin(which is inactive or rendered inactive, in order to avoid agonisticRANKL binding to its receptor RANK), receptor-fusion protein, e.g. aRANK-Fc fusion protein, a peptide, aptamer, or a small molecule.

Methods for producing and characterizing an antagonistic agent arewell-known in the art. In a preferred embodiment, antagonistic bindersare produced and screened for predefined properties using one or morecell-based assays. Such assays often involve monitoring the response ofcells to a binder, for example cell survival, cell death, change incellular morphology, or transcriptional activation such as cellularexpression of a natural gene or reporter gene.

The production of the recombinant polypeptide antagonistic agentpreferably employs an expression system to produce the recombinantpolypeptide, e.g. including expression constructs or vectors comprisinga nucleotide sequence encoding the polypeptide.

In one embodiment, the antagonistic agent is identified through a drugdiscovery process, such as including a screen employing combinatoriallibraries (random or semi-random) containing potential drug candidates,e.g. peptide libraries, antibody libraries, or chemical compoundlibraries. Screens may be performed in a high throughput manner usinge.g. flow cytometry, and optionally can discriminate between active andnon-active or blocked RANK/RANKL interaction. Biological screens may aimat finding novel antagonistic agents specifically targeting pRANKL.

By “substantially reducing or inhibiting” the RANK-RANKL signalling, itis meant that the antagonistic agent (i) inhibits the binding of RANKLto RANK by more than 50%, preferably more than 60%, 70%, 80%, 90% or95%, or completely inhibits such binding; and/or (ii) functionallyinhibits the RANKL-induced pathway, and in particular the signallingfollowing RANK stimulation, e.g. activities of MAPK (Mitogen-ActivatedProtein Kinase) or SRC-Kinases, or NF-κB signals involved in metastasisformation, e.g. as determined in a lung model as described in theexamples section below. Such functional inhibition is e.g. inhibitingmetastasis formation by more than about 50%, 60%, 70%, 80%, 90% or 95%,or complete inhibition.

Alternatively, the functional inhibition may be determined ex vivo, e.g.determining the migration of cancer cells via cytoskeletalrearrangements brought on by the activation of Erk1/2 and Src in astandard assay. Specifically, migration and invasion potential of tumorcells may be measured by determining the portion of cells that havepassed a porous and/or extracellular-matrix mimicking barrier. Suchfunctional inhibition is e.g. inhibiting migration and/or invasion bymore than about 50%, 60%, 70%, 80%, 90% or 95%, or complete inhibition.

The functional inhibition may also be determined by measuring thedownregulation of a epithelial-mesenchymal transition (EMT) genesignature, in particular metastasis-associated genes in cancer cells bytargeting pRANKL, e.g. by quantitative PCR-based methods, determiningany of E-Cadherin, Claudin, SNAIL, or Fibronectin.

The term “antibody” as used herein shall refer to polypeptides orproteins that consist of or comprise antibody domains, which areunderstood as constant and/or variable domains of the heavy and/or lightchains of immunoglobulins, with or without a linker sequence. Theantibody as used herein has a specific antigen-binding site to bind theRANKL antigen or one or more epitopes of such antigen, specificallycomprising a CDR binding site of a single variable antibody domain, suchas VH, VL or VHH, or a binding site of pairs of variable antibodydomains, such as a VL/VH pair, an antibody comprising a VL/VH domainpair and constant antibody domains, such as Fab, F(ab′), (Fab)₂, scFv,Fv, or a full length antibody.

Specific antibody formats may be used according to the invention, e.g.an antibody comprising or consisting of single variable antibody domain,such as VH, VL or VHH, or combinations of variable and/or constantantibody domains with or without a linking sequence or hinge region,including pairs of variable antibody domains, such as a VL/VH pair, anantibody comprising or consisting of a VL/VH domain pair and constantantibody domains, such as heavy-chain antibodies, Fab, F(ab′), (Fab)₂,scFv, Fd, Fv, or a full-length antibody, e.g. of an IgG type (e.g., anIgG1, IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE, or IgMantibody. The term “full length antibody” can be used to refer to anyantibody molecule comprising at least most of the Fc domain and otherdomains commonly found in a naturally occurring antibody monomer. Thisphrase is used herein to emphasize that a particular antibody moleculeis not an antibody fragment.

The term “antibody” shall specifically include antibodies in theisolated form, which are substantially free of other antibodies directedagainst different target antigens or comprising a different structuralarrangement of antibody domains. Still, an isolated antibody may becomprised in a combination preparation, containing a combination of theisolated antibody, e.g. with at least one other antibody, such asmonoclonal antibodies or antibody fragments having differentspecificities.

The term “antibody” shall apply to antibodies of animal origin,including human species, such as mammalian, such as human or murine, oravian, such as hen, which term shall particularly include recombinantantibodies that are based on a sequence of animal origin, e.g. humansequences.

The term “antibody” further applies to chimeric antibodies withsequences of origin of different species, such as sequences of murineand human origin.

The term “chimeric” as used with respect to an antibody refers to thoseantibodies wherein one portion of each of the amino acid sequences ofheavy and light chains is homologous to corresponding sequences inantibodies derived from a particular species or belonging to aparticular class, while the remaining segment of the chain is homologousto corresponding sequences in another species or class. Typically thevariable region of both light and heavy chains mimics the variableregions of antibodies derived from one species of mammals, while theconstant portions are homologous to sequences of antibodies derived fromanother. For example, the variable region can be derived from presentlyknown sources using readily available B-cells or hybridomas fromnon-human host organisms in combination with constant regions derivedfrom, for example, human cell preparations.

The term “antibody” may further apply to humanized antibodies.

The term “humanized” as used with respect to an antibody refers to amolecule having an antigen binding site that is substantially derivedfrom an immunoglobulin from a non-human species, wherein the remainingimmunoglobulin structure of the molecule is based upon the structureand/or sequence of a human immunoglobulin. The antigen binding site mayeither comprise complete variable domains fused onto constant domains oronly the complementarity determining regions (CDR) grafted ontoappropriate framework regions (FR) in the variable domains.Antigen-binding sites may be wild-type or modified, e.g. by one or moreamino acid substitutions, preferably modified to resemble humanimmunoglobulins more closely. Some forms of humanized antibodiespreserve all CDR sequences (for example a humanized mouse antibody whichcontains all six CDRs from the mouse antibody). Other forms have one ormore CDRs which are altered with respect to the original antibody.

The term “antibody” further applies to human antibodies.

The term “human” as used with respect to an antibody, is understood toinclude antibodies having variable and constant regions derived fromhuman germline immunoglobulin sequences. The human antibody of theinvention may include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo), forexample in the CDRs. Human antibodies include antibodies isolated fromhuman immunoglobulin libraries or from animals transgenic for one ormore human immunoglobulin.

The term “antibody” specifically applies to antibodies of any class orsubclass. Depending on the amino acid sequence of the constant domain oftheir heavy chains, antibodies can be assigned to the major classes ofantibodies IgA, IgD, IgE, IgG, and IgM, and several of these may befurther divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3,IgG4, IgA1, and IgA2.

The term further applies to monoclonal or polyclonal antibodies,specifically a recombinant antibody, which term includes all antibodiesand antibody structures that are prepared, expressed, created orisolated by recombinant means, such as antibodies originating fromanimals, e.g. mammalians including human, that comprises genes orsequences from different origin, e.g. murine, chimeric, humanizedantibodies, or hybridoma derived antibodies. Further examples refer toantibodies isolated from a host cell transformed to express theantibody, or antibodies isolated from a recombinant, combinatoriallibrary of antibodies or antibody domains, or antibodies prepared,expressed, created or isolated by any other means that involve splicingof antibody gene sequences to other DNA sequences.

Antibody domains may be of native structure or modified by mutagenesisor derivatisation, e.g. to modify the antigen binding properties or anyother property, such as stability or functional properties, such asbinding to the Fc receptors FcRn and/or Fcgamma receptor (FCGR).Polypeptide sequences are considered to be antibody domains, ifcomprising a beta-barrel structure consisting of at least twobeta-strands of an antibody domain structure connected by a loopsequence.

It is understood that the term “antibody” also refers to derivatives ofan antibody, in particular functionally active derivatives, herein alsoreferred to as functional variants of antibodies. An antibody derivativeis understood as any combination of one or more antibody domains orantibodies and/or a fusion protein, in which any domain of the antibodymay be fused at any position of one or more other proteins, such asother antibodies, e.g. a binding structure comprising CDR loops, areceptor polypeptide, but also ligands, scaffold proteins, enzymes,toxins and the like. A derivative of the antibody may be obtained byassociation or binding to other substances by various chemicaltechniques such as covalent coupling, electrostatic interaction,di-sulfide bonding etc. The other substances bound to the antibody maybe lipids, carbohydrates, nucleic acids, organic and inorganic moleculesor any combination thereof (e.g. PEG, prodrugs or drugs). In a specificembodiment, the antibody is a derivative comprising a drug, e.g. toobtain an antibody-drug conjugate. Specifically, the antibody may beused together with a tag. Thus, the antibody may be a derivativecomprising a tag, such as for analytical or diagnostic purposes,including e.g. for use as in vivo diagnostic. There is not a specificlimitation with respect to the usable tag, as far as it has no ortolerable negative impact on the binding of the antibody to its targetantigen. Examples of suitable tags include His-tag, Myc-tag, FLAG-tag,Strep-tag, Calmodulin-tag, GST-tag, MBP-tag, and S-tag. In anotherspecific embodiment, the antibody is a derivative comprising a label.The term “label” as used herein refers to a detectable compound orcomposition which is conjugated directly or indirectly to the antibodyso as to generate a “labeled” antibody. The label may be detectable byitself, e.g. radioisotope labels or fluorescent labels, or, in the caseof an enzymatic label, may catalyze chemical alteration of a substratecompound or composition which is detectable.

The term derivative also includes fragments, variants, analogs orhomologs of antibodies, e.g. with a specific glycosylation pattern, e.g.produced by glycoengineering, which are functional and may serve asfunctional variants, e.g. binding to the specific target.

The term “glycoengineered” with respect to antibody sequences shallrefer to glycosylation variants having modified immunogenic properties,ADCC and/or CDC as a result of the glycoengineering. All antibodiescontain carbohydrate structures at conserved positions in the heavychain constant regions, with each isotype possessing a distinct array ofN-linked carbohydrate structures, which variably affect proteinassembly, secretion or functional activity. IgG1 type antibodies areglycoproteins that have a conserved N linked glycosylation site atAsn297 in each CH2 domain. The two complex bi-antennary oligosaccharidesattached to Asn297 are buried between the CH2 domains, forming extensivecontacts with the polypeptide backbone, and their presence is essentialfor the antibody to mediate effector functions such as antibodydependent cellular cytotoxicity (ADCC). Removal of N-Glycan at N297,e.g. through mutating N297, e.g. to A, or T299 typically results inaglycosylated antibodies with reduced ADCC.

Major differences in antibody glycosylation occur between cell lines,and even minor differences are seen for a given cell line grown underdifferent culture conditions. Expression in bacterial cells typicallyprovides for an aglycosylated antibody.

Antibodies can be devoid of an active Fc moiety, thus, either composedof antibody domains that do not have an FCGR binding site, specificallyincluding any antibody devoid of a chain of CH2 and CH3 domains, orcomprising antibody domains lacking Fc effector function, e.g. bymodifications to reduce Fc effector functions, in particular to abrogateor reduce ADCC and/or CDC activity. Such modifications may be effectedby mutagenesis, e.g. mutations in the FCGR binding site or byderivatives or agents to interfere with ADCC and/or CDC activity of anantibody, so to achieve reduction of Fc effector function or lack of Fceffector function, which is typically understood to refer to Fc effectorfunction of less than 10% of the unmodified (wild-type) antibody,preferably less than 5%, as measured by ADCC and/or CDC activity.

An antibody of the present invention may or may not exhibit Fc effectorfunction. Though the mode of action is mainly mediated by inhibiting theRANKL-RANK signaling in the tumor cell microenvironment, without Fceffector functions, Fc can recruit complement and aid elimination of thetarget platelet-tumor cell aggregates, from the circulation viaformation of immune complexes.

Exemplary antibodies comprise an Fc fragment or at least part of an Fcfragment, such as to maintain the binding site to FcRn, therebyobtaining an antibody with substantive half-life in vivo.

A further example refers to modification of an Fc to obtain reduction ofpossible ADCC and/or CDC activity, e.g. by a switch of IgG1 to IgG2subtype or by modifications to reduce binding to the Fc receptor, e.g.by E233P and/or L234V and/or L235A and/or D265G and/or A327Q and/orA330A and/or G236, deletion and/or A327G and/or A330S in a human IgG1Fc, wherein numbering is according to Kabat [EU-Index].

Further examples refer to a modification to reduce immunogenicity, e.g.by a K. O. glycosylation site, such as N297Q, which provides for animpaired binding to lectin receptor.

An exemplary antibody is Denosumab, or a functional variant or anantigen-binding fragment thereof, e.g. incorporated in the framework ofan IgG2 or any other immunoglobulin types or subtypes. For example, theDenosumab antigen-binding site or CDR sequences may be incorporated intoan IgG1 antibody, with or without Fc effector function.

It is understood that the term “antibody” also refers to variants of anantibody, including antibodies with functionally active CDR variants ofa parent CDR sequence, and functionally active variant antibodies of aparent antibody. For example, functional variants of Denosumab may beengineered and used as further described herein.

Specifically, an antibody variant derived from an antibody of theinvention may comprise at least one or more of the CDR regions or CDRvariants thereof (of the parent antibody), e.g. at least 3 CDRs of theheavy chain variable region and/or at least 3 CDRs of the light chainvariable region, with at least one point mutation in at least one of theCDR or FR regions, or in the constant region of the heavy chain (HC) orlight chain (LC), being functionally active, e.g. specifically bindingthe RANKL antigen.

The term “variant” shall particularly refer to antibodies, such asmutant antibodies or fragments of antibodies, e.g. obtained bymutagenesis methods, in particular to delete, exchange, introduceinserts into a specific antibody amino acid sequence or region orchemically derivatise an amino acid sequence, e.g. in the constantdomains to engineer the antibody stability, effector function orhalf-life, or in the variable domains to improve antigen-bindingproperties, e.g. by affinity maturation techniques available in the art.Any of the known mutagenesis methods may be employed, including pointmutations at desired positions, e.g. obtained by randomizationtechniques. In some cases positions are chosen randomly, e.g. witheither any of the possible amino acids or a selection of preferred aminoacids to randomize the antibody sequences. The term “mutagenesis” refersto any art recognized technique for altering a polynucleotide orpolypeptide sequence. Preferred types of mutagenesis include error pronePCR mutagenesis, saturation mutagenesis, or other site directedmutagenesis.

The term “functionally active variant” of an antibody means a sequenceresulting from modification of this sequence (a parent antibody or aparent sequence) by insertion, deletion or substitution of one or moreamino acids, or chemical derivatisation of one or more amino acidresidues in the amino acid sequence, or nucleotides within thenucleotide sequence, or at either or both of the distal ends of thesequence, e.g. in a CDR sequence the N-terminal and/or C-terminal 1, 2,3, or 4 amino acids, and/or the centric 1, 2, 3, or 4 amino acids (i.e.in the midst of the CDR sequence), and which modification does notaffect, in particular impair, the activity of this sequence. In the caseof a binding site having specificity to a selected target antigen, thefunctionally active variant of an antibody would still have thepredetermined binding specificity, or substantially the same biologicalactivity, though this could be changed, e.g. to change the finespecificity to a specific epitope, the affinity, the avidity, the Kon orKoff rate, etc. For example, an affinity matured antibody isspecifically understood as a functionally active variant antibody.Hence, the modified CDR sequence in an affinity matured antibody isunderstood as a functionally active CDR variant.

Preferably, an agent is used which binds to pRANKL with a high affinity,in particular with a high on and/or a low off rate, or a high avidity ofbinding. The binding affinity is usually characterized in terms of theconcentration of the agent, at which half of the binding sites areoccupied, known as the dissociation constant (Kd, or KD). Usually abinder is considered a high affinity binder with a Kd<10⁻⁸ M, preferablya Kd<10⁻⁹ M, even more preferred is a Kd<10⁻¹⁰ M.

Yet, in an alternatively preferred embodiment the individual antigenbinding affinities are of medium affinity, e.g. with a Kd of less than10⁻⁶ M and up to 10⁻⁸ M, e.g. when binding to at least two antigens.

The term “substantially the same biological activity” as used hereinrefers to the activity as indicated by substantially the same activitybeing at least 20%, at least 50%, at least 75%, at least 90%, e.g. atleast 100%, or at least 125%, or at least 150%, or at least 175%, ore.g. up to 200% of the activity as determined for the comparable orparent antibody.

In a preferred embodiment the functionally active variant of a parentantibody

a) is a biologically active fragment of the antibody, the fragmentcomprising at least 50% of the sequence of the molecule, preferably atleast 60%, at least 70%, at least 80%, at least 90%, or at least 95% andmost preferably at least 97%, 98% or 99%;

b) is derived from the antibody by at least one amino acid substitution,addition and/or deletion, wherein the functionally active variant has asequence identity to the molecule or part of it, such as an antibody ofat least 50% sequence identity, preferably at least 60%, more preferablyat least 70%, more preferably at least 80%, still more preferably atleast 90%, even more preferably at least 95% and most preferably atleast 97%, 98% or 99%; and/or

c) consists of the antibody or a functionally active variant thereof andadditionally at least one amino acid or nucleotide heterologous to thepolypeptide or the nucleotide sequence.

In one preferred embodiment of the invention, the functionally activevariant of the antibody according to the invention is essentiallyidentical to the variant described above, but differs from itspolypeptide or the nucleotide sequence, respectively, in that it isderived from a homologous sequence of a different species. These arereferred to as naturally occurring variants or analogs.

The term “functionally active variant” also includes naturally occurringallelic variants, as well as mutants or any other non-naturallyoccurring variants. As is known in the art, an allelic variant is analternate form of a (poly)peptide that is characterized as having asubstitution, deletion, or addition of one or more amino acids that doesessentially not alter the biological function of the polypeptide.

Functionally active variants may be obtained by sequence alterations inthe polypeptide or the nucleotide sequence, e.g. by one or more pointmutations, wherein the sequence alterations retains or improves afunction of the unaltered polypeptide or the nucleotide sequence, whenused in combination of the invention. Such sequence alterations caninclude, but are not limited to, (conservative) substitutions,additions, deletions, mutations and insertions.

Conservative substitutions are those that take place within a family ofamino acids that are related in their side chains and chemicalproperties. Examples of such families are amino acids with basic sidechains, with acidic side chains, with non-polar aliphatic side chains,with non-polar aromatic side chains, with uncharged polar side chains,with small side chains, with large side chains etc.

A point mutation is particularly understood as the engineering of apolynucleotide that results in the expression of an amino acid sequencethat differs from the non-engineered amino acid sequence in thesubstitution or exchange, deletion or insertion of one or more single(non-consecutive) or doublets of amino acids for different amino acids.

Preferred point mutations refer to the exchange of amino acids of thesame polarity and/or charge. In this regard, amino acids refer to twentynaturally occurring amino acids encoded by sixty-four triplet codons.These 20 amino acids can be split into those that have neutral charges,positive charges, and negative charges:

The “neutral” amino acids are shown below along with their respectivethree-letter and single-letter code and polarity:

Alanine: (Ala, A) nonpolar, neutral;

Asparagine: (Asn, N) polar, neutral;

Cysteine: (Cys, C) nonpolar, neutral;

Glutamine: (Gln, Q) polar, neutral;

Glycine: (Gly, G) nonpolar, neutral;

Isoleucine: (Ile, I) nonpolar, neutral;

Leucine: (Leu, L) nonpolar, neutral;

Methionine: (Met, M) nonpolar, neutral;

Phenylalanine: (Phe, F) nonpolar, neutral;

Proline: (Pro, P) nonpolar, neutral;

Serine: (Ser, S) polar, neutral;

Threonine: (Thr, T) polar, neutral;

Tryptophan: (Trp, W) nonpolar, neutral;

Tyrosine: (Tyr, Y) polar, neutral;

Valine: (Val, V) nonpolar, neutral; and

Histidine: (His, H) polar, positive (10%) neutral (90%).

The “positively” charged amino acids are:

Arginine: (Arg, R) polar, positive; and

Lysine: (Lys, K) polar, positive.

The “negatively” charged amino acids are:

Aspartic acid: (Asp, D) polar, negative; and

Glutamic acid: (Glu, E) polar, negative.

“Percent (%) amino acid sequence identity” with respect to the antibodysequences and homologs described herein is defined as the percentage ofamino acid residues in a candidate sequence that are identical with theamino acid residues in the specific polypeptide sequence, after aligningthe sequence and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. Those skilled in the artcan determine appropriate parameters for measuring alignment, includingany algorithms needed to achieve maximal alignment over the full lengthof the sequences being compared.

The term “antigen” as used herein interchangeably with the terms“target” or “target antigen” shall refer to a whole target molecule or afragment of such molecule recognized by an agent specificallyrecognizing the antigen, or capable of specifically binding the target,such as an antibody which recognizes the antigen through binding by theantibody binding site. Specifically, substructures of an antigen, e.g. apolypeptide or carbohydrate structure, generally referred to as“epitopes”, e.g. B-cell epitopes or T-cell epitope, which areimmunologically relevant, may be recognized by such binding site. Theterm “epitope” as used herein shall in particular refer to a molecularstructure which may completely make up a specific binding partner or bepart of a specific binding partner to a binding site of an agent asdescribed herein. An epitope may either be composed of a carbohydrate, apeptidic structure, a fatty acid, an organic, biochemical or inorganicsubstance or derivatives thereof and any combinations thereof. If anepitope is comprised in a peptidic structure, such as a peptide, apolypeptide or a protein, it will usually include at least 3 aminoacids, preferably 5 to 40 amino acids, and more preferably between about10-20 amino acids. Epitopes can be either linear or conformationalepitopes. A linear epitope is comprised of a single segment of a primarysequence of a polypeptide or carbohydrate chain. Linear epitopes can becontiguous or overlapping. Conformational epitopes are comprised ofamino acids or carbohydrates brought together by folding the polypeptideto form a tertiary structure and the amino acids are not necessarilyadjacent to one another in the linear sequence.

The antigen as described herein is human RANKL, in particular pRANKL,though the specific binder to pRANKL may cross-specifically recognizesRANKL and/or mRANKL, or be cross-reactive with pRANKL and any of (orboth of), sRANKL and mRANKL. The human pRANKL is specifically understoodas RANKL originating from human blood platelets (also referred to asthrombocytes), e.g. an antigen expressed on the surface of a human bloodplatelet, preferably by an activated platelet, which can be targetedwith an antagonist that binds thereto. The platelet can also interactwith a (RANKL negative) cancer cell to transform such cancer cell into apremetastatic lesion, which itself is capable of expressing RANKL. Thus,the antagonistic agent may as well target the cancer cell expressingRANKL. The pRANKL may be detached or shedded from the surface of theplatelet and inhibited by the antagonistic agent as a soluble ligand.pRANKL when expressed on the surface of platelets, may differ fromsRANKL in the accessibility of the epitopes. mRANKL may be expressed bytissue, or a cancer cell, and further interacting with a platelet and/oranother cancer cell. Further structural differences of pRANKL ascompared to other forms of RANKL or compared to RANKL originating formcancer cells and cells of other origin like osteoblasts may be evidentupon thorough analysis of the amino acid sequence and glycosylationpattern of pRANKL.

The term “RANKL” includes any variants, isoforms and species homologs ofhuman RANKL which are naturally expressed by cells and which are boundto the surface of cells, e.g. of human blood platelets or tumor cells,or which are present as soluble RANKL in the circulation, as determinedin a sample of peripheral blood.

Preferred epitopes of pRANKL are incorporated in the extracellularportion of the RANKL antigen, in particular the extracellular part ofthe pRANKL or the extracellular part of the transmembrane RANKL, e.g. anepitope which is accessible on the surface of the platelets or cells.

The antagonistic agent as described herein is binding to an epitope ofRANKL, which leads to substantial inhibition of the RANKL binding to itsreceptor RANK, thereby inhibiting the signalling pathway. Since RANKLpromotes survival and induces migration of various cancer cells thatexpress RANK, the antagonistic agent as described herein would interferewith the proliferation and metastasis of cancer cells by preventing orreducing premetastatic migration and aggregation of cancer or tumorcells.

The term “metastasis” as described herein shall refer to the spread ofmalignant tumors to secondary sites, e.g. remote from an original orprimary tumor. This normally involves detachment of cancer cells from aprimary tumor, entering the body circulation and settling down to growwithin normal tissues elsewhere in the body. Such primary tumor isunderstood as a tumor growing at the site of the cancer origin.Hematopoietic diseases (leukemia, lymphomas and myeloma) are considereddisseminated at time of diagnosis. However, also hematopoietic cancercan form metastatic tumors. Although rare, the metastasis of blood andlymphatic system cancers to the lung, heart, central nervous system, andother tissues has been reported. Metastatic tumor cells are understoodas cells that have the ability to produce a metastasis or are already apart of a metastatic tumor. Specifically, the primary cancer cellsand/or the metastasis as referred to herein is RANK-positive, e.g. asdetermined by a standard immunohistochemical or a PCR-based method.

Examples of primary mesenchymal tumors are soft tissue tumors, e.g.deriving from muscle, fibrous tissue, and vascular tissue.

Among primary mesodermal and/or ectodermal tumors are melanoma and/orameloblastoma and primitive neuro-ectodermal tumor of the lung,respectively.

Representative primary, epithelial cell cancers include amongst othersbreast, prostate, lung, bladder, uterine, ovarian, brain, head and neck,esophageal, pancreatic, gastric, germ cell, and colorectal cancers.

Particular important RANK-positive tumor diseases are breast cancer,pancreatic cancer, gastric cancer, esophageal cancer, renal cellcarcinoma, lung carcinoma, colon/rectal/colorectal cancer, melanoma,prostate cancer, head and neck cancer, or other diseases associated withRANK-positive tumor entities.

Among the blood cancers are leukemia, lymphoma, or myeloma.

A patient suffering from leukemia can specifically benefit from theanti-RANKL treatment as described herein, because RANK signaling intoleukemic cells may e.g. enhance their proliferative potential and/oralter their resistance to anti-cancer therapeutic intervention e.g. withchemotherapy and/or kinase inhibitors.

Patients treated for cancer and primary tumors often retain a minimalresidual disease related to the cancer. That is, even though a patientmay have by clinical measures a complete remission of the disease inresponse to treatment, a small fraction of the cancer cells may remainthat have escaped destruction. The type and size of this residualpopulation is an important prognostic factor for the patient's continuedtreatment.

In certain embodiments, the patient has minimal residual disease afterthe primary cancer therapy (e.g. chemotherapy, radiation therapy and/orsurgery). The antagonistic agent as described herein would beparticularly combined with cytoreductive therapy or other therapeuticinterventions e.g. immunotherapy, to treat minimal residual disease,and/or as maintenance therapy, e.g. as a prolonged or extended therapyafter cessation of another cancer treatment. In addition, theantagonistic agent would delay the re-growth or recurrence of the canceror tumor, or recurrence of metastasis formation in metastatic disease,e.g. by at least 1 or more months.

Specific metastatic tumor cells are disseminating tumor cells which tendto develop premetastatic tumor cell aggregates in the circulation thattrigger metastasis formation in distant organs. It surprisingly tunedout that disseminating RANK-positive tumor cells can aggregate withRANKL-positive platelets in the circulation, such aggregates inducingmetastasis formation. Activated platelets can upregulate pRANKL andthereby stimulate RANK on the tumor cells and thus render thempremetastatic. Therefore, the cancer patient advantageously treated asdescribed herein suffers from a tumor disease or cancer which mainlymetastasizes via the blood vessels, and not or less through thelymphatics.

The platelet-cancer cell aggregates are understood as prometastatic, ora preform of metastases, in particular upon activation of the plateletsto express pRANKL. Such preform differs from metastases because theaggregates are present in the circulation, and not yet growing to largermass as metastases in distant organs. To this point, the prometastaticplatelet-cancer cell aggregates are considered an embodiment of aRANK-positive neoplastic disease.

The term “disseminating tumor cells” as used herein primarily refers totumor cells found in circulation of a patient having a tumor. Thoughthis term typically would not include hematological tumors where themajority of the tumor is found in circulation, the term “Disseminatingtumor cell” is as well encompassing (pre)metastatic tumor cells in apatient suffering from blood cancer. Blood cancers may trigger contactof platelets with the cancer cells, optionally resulting indisseminating platelet-cancer cell contact aggregates in the circulationonce they are going to metastasize. Blood cancer cells acquire theability to penetrate the walls of lymphatic or blood vessels, afterwhich they are able to circulate through the bloodstream as circulating(disseminating) tumor cells to other sites and tissues in the body,eventually forming a clinically detectable tumor known as a metastaticor secondary tumor implant. In addition, the pRANKL-RANK signalling maytrigger blood cancer disease progression.

The term “prometastatic” as used herein in relation to platelet-cancercell aggregates is understood in the following way. Cancer or tumorcells and/or platelets can be prometastatic, i.e. promoting metastasis,because of the tendency of aggregating and subsequently binding of thepRANKL-positive platelets to the RANK-positive tumor cell, andinitiating the RANK-RANKL signalling. Reciprocal interactions betweenthe cancer cells and the various components of the tumormicroenvironment influence tumor progression and metastases although themolecular mechanisms underlying these metastasis-promoting effects areyet ill defined. Identifying and understanding pathways ofcancer-platelet or tumor-platelet cross-talk can lead to the developmentof therapies targeting pRANKL to prevent metastasis at its earlieststage, resulting in improved patient outcome.

“Premetastatic lesions” are herein understood as a precursor lesion,characterized by early cellular and molecular events of cancerdissemination that lead to the creation of a metastasis-promotingmicroenvironment (pre-metastatic niches). It surprisingly turned outthat disseminating tumor cells would cause metastasis upon recruitmentof thrombocytes, in particular activated thrombocytes expressing pRANKL.Through interaction with the blood platelets, optionally formingdetectable (prometastatic) platelet-cancer cells aggregates, and uponRANK-RANKL signaling in the microenvironment, the cancer cell wouldbecome pre-metastatic, thus change its appearance or nature before itbecomes metastatic. Thus, the pRANKL is considered characteristicdistinguishing between those cells that are premetastatic lesions ornot. The pRANKL is therefore a new target of metastasis prevention orapproaches to detect and prevent metastasis at its earliest inception.

By reducing the prometastatic platelet-cancer cell aggregates, the riskof organ-specific metastasis formation is greatly reduced. Inparticular, this refers to visceral or mesothelial metastasis, or targetorgans like liver, lung, bone, intestine, skin, muscle, spleen, brain,or kidney, and in many cases target sites other than bones. Visceralmetastasis in particular refers to metastases in the viscera, theinternal organs of the body, specifically those within the chest such asheart or lungs or the abdomen, such as the liver, pancreas orintestines. Mesothelial metastasis refers to the growth of cancer cellsin or at a mesothelium such as the pleura and the peritoneum. Inparticular, mesothelial metastases can lead to accumulation of fluid inthe cavity surrounded by the mesothelium, in particular pleural and/orabdominal effusion, e.g. due to inflammatory reaction and/or increasedpermeability of the affected mesothelium caused by the metastases.

The term “metastatic potential” as used herein shall refer to thepotential for developing minimal residual disease, the recurrence ofmetastatic disease, the potential for metastatic cancer to progressrapidly, or the potential for metastatic cancer to display resistance toa standard therapy, e.g. chemotherapy and/or immunotherapy.

An increased or high metastatic potential is indicative of a propensityto form distant metastasis or metastasis to multiple sites or organs, orelse local, tissue specific, organ-specific, or site-specificmetastasis. A high metastatic potential is indicated in a cancerpatient, where a relatively high load of disseminated tumor cells aredetermined in the circulation. In particular, the peripheral bloodsample of a cancer patient would contain an increased number ofdetectable platelets expressing pRANKL, or any of sRANKL or mRANKL, orconglomerates of cancer cells and platelets, as compared to a referencevalue. Examples for cell lines of high metastatic potential areMDA-MB-231 (mamma carcinoma cell line, derived from metastatic site ofpatient with Her2/neu positive breast cancer, e.g. available at ATCC(Manassas, Va.)), or SK-Mel-5 (melanoma cell line, derived frommetastatic site of a patient with malignant melanoma, e.g. available atATCC (Manassas, Va.)).

In contrast, a low metastatic potential is indicative of a low rate ofmetastasis or a non-metastatic tumor. The RANK-positive cancer of lowmetastatic potential would present only with low or limited amount oftumor cells in the circulation. In particular, the peripheral bloodsample of a cancer patient would contain only a low or limited number ofdetectable platelets expressing pRANKL, or pRANKL, or conglomerates oftumor cells and platelets. Examples for cell lines of low metastaticpotential are MCF-7 (mamma carcinoma cell line, derived from metastaticsite of a patient with Her2/neu positive breast cancer, e.g. availableat ATCC (Manassas, Va.)), or SK-BR-3 (mamma carcinoma cell line, derivedfrom metastatic site of a patient with Her2/neu positive breast cancer,e.g. available at ATCC (Manassas, Va.)).

Methods of analyzing disseminating tumor cells and assessing theirmetastatic potential in vivo and in vitro are well-known in the art.Such methods can be improved by the specific determination of pRANKLexpression, or by determining the level of premetastatic lesions, inparticular prometastatic platelet-cancer cell aggregates in a bloodsample, or a platelet containing fraction thereof.

The prognostic assay based on the method of predicting the metastaticpotential as further described herein, can be used to determine whethera patient is suitably treated with an agent, e.g. an agonist,antagonist, peptidomimetic, protein, peptide, nucleic acid, smallmolecule, or other drug candidate to treat cancer or other disordersassociated with cancer such as metastatic disease. For example, suchassay can be used to determine whether a subject shall be administeredwith a chemotherapeutic agent.

The term “patient” as used herein shall refer to a warm-bloodedmammalian, particularly a human being. In particular the medical useformat of the invention or the respective method of treatment applies toa patient in need of prophylaxis or treatment of cancer, tumor ormetastatic disease. The patient may be suffering from early stage orlate stage disease, or else a patient predisposed of such disease, e.g.by genetic predisposition.

The term “pharmaceutical composition” as described herein shall refer toa composition suitable for administering to a human, i.e. a compositioncontaining components which are pharmaceutically acceptable. Preferably,a pharmaceutical composition comprises an active compound or a saltthereof together with a carrier, diluent or pharmaceutical excipientsuch as buffer, or tonicity modifier.

The antagonistic agent of the present invention is specifically providedin a pharmaceutical composition. Stable pharmaceutical compositions arecontemplated which are prepared for storage. In specific embodiments,the agent having the desired degree of purity is mixed withpharmaceutically acceptable carriers, excipients or stabilizers, andprovided as lyophilized formulation, aqueous solution or oil-in-wateremulsion. Typically such compositions comprise a pharmaceuticallyacceptable carrier as known and called for by acceptable pharmaceuticalpractice, see e.g. Remingtons Pharmaceutical Sciences, 16th edition(1980) Mack Publishing Co. Examples of such carriers include sterilizedcarriers such as saline, Ringers solution or dextrose solution,optionally buffered with suitable buffers to a pH within a range of 5 to8.

The formulations to be used for in vivo administration will need to besterile. This is readily accomplished by filtration through sterilefiltration membranes or other suitable methods.

Administration of the pharmaceutical composition comprising the agentfor use as described herein is specifically by the systemic route or byparenteral administration, e.g. by the intravenous, intramuscular orsubcutaneous route, but also orally, intranasally, intraotically,transdermally, mucosal, topically (e.g., gels, salves, lotions, creams,etc.), intraperitoneally, intramuscularly, intrapulmonary, vaginally,parenterally, rectally or intraocularly. Exemplary formulations as usedfor parenteral administration include those suitable for intravenous,intramuscular, or subcutaneous injection as, for example, a sterilesolution or suspension.

In particular, the intravenous administration is preferred, e.g. asintravenous infusion or as a bolus injection. Denosumab is known to beadministered by the subcutaneous route. In the new indication oftargeting pRANKL, the Denosumab agent would specifically be administeredsuch that it is available in the circulation for a prolonged period oftime, thus, the subcutaneous route is specifically less preferred oravoided.

The present invention includes a pharmaceutical preparation, containingas active substance the antagonistic agent in a therapeuticallyeffective amount.

The term “therapeutically effective amount”, used herein interchangeablywith any of the terms “effective amount” or “sufficient amount” of theantagonistic agent as described herein, is a quantity or activitysufficient to, when administered to the subject effect beneficial ordesired results, including clinical results, and, as such, an effectiveamount or synonym thereof depends upon the context in which it is beingapplied. In the context of disease, therapeutically effective amounts ofthe agent may be used to treat, modulate, attenuate, reverse, or affecta disease or condition that benefits from a down-regulation or reductionof premetastatic lesions, platelet-cancer cell aggregates, orprometastatic tumor cell aggregates, e.g. for preventing or treatingmetastatic disease. An effective amount is intended to mean that amountof a compound that is sufficient to treat, prevent or inhibit suchdiseases or disorder. The amount of the antagonistic agent that willcorrespond to such an amount will vary depending on various factors,such as the given drug or compound, the pharmaceutical formulation, theroute of administration, the type of disease or disorder, the identityof the subject or host being treated, and the like, but can neverthelessbe routinely determined by one skilled in the art.

Moreover, a treatment or prevention regime of a subject (a cancerpatient) with a therapeutically effective amount of the antagonisticagent may consist of a single administration, or alternatively comprisea series of applications. For example, the antagonistic agent may beadministered at least once a year, at least once a half-year or at leastonce a month. However, in another embodiment, the antagonistic agent maybe administered to the subject from about one time per week to about adaily administration for a given treatment. The length of the treatmentperiod depends on a variety of factors, such as the severity of thedisease, the age of the patient, the concentration and the activity ofthe antagonistic agent. It will also be appreciated that the effectivedosage used for the treatment or prophylaxis may increase or decreaseover the course of a particular treatment or prophylaxis regime. Changesin dosage may result and become apparent by standard diagnostic assaysknown in the art. In some instances, chronic administration may berequired.

A therapeutically effective amount of the antagonistic agent such asprovided to a human patient in need thereof may specifically be in therange of 0.5-1000 mg, preferably 1-400 mg, even more preferred up to 300mg, up to 200 mg, up to 100 mg or up to 10 mg, though higher doses maybe indicated e.g. for treating acute disease conditions, such as whenpreparing for a surgical intervention, or shortly after a surgicalintervention, when starting treatment within a 1-7 days followingsurgery. Subcutaneous doses typically are. ranging within 0.5 and 400mg.

The term “treatment” as used herein shall always refer to treatingpatients for prophylactic (i.e. to prevent a disease or diseasecondition) or therapeutic (i.e. to treat a disease or disease condition)purposes. Treatment of a patient will typically be therapeutic in casesof cancer. However, in case of patients suffering from a primarydisease, which are at risk of disease progression or at risk ofdeveloping a secondary disease condition or side reaction, e.g. which isdependent on the RANK-RANKL signalling effects, the treatment may beprophylactic. Such treatment for prophylaxis is herein also referred toas treatment or therapy, e.g. employing a therapeutically effectiveamount.

In one embodiment, the antagonistic agent is the only therapeuticallyactive agent administered to a patient, e.g. as a disease modifyingmonotherapy.

Alternatively, the antagonistic agent is administered in combinationwith one or more other therapeutic agents, including but not limited tostandard treatment, e.g. chemotherapeutics to treat malignant disease.

In a combination therapy, the antagonistic agent may be administered asa mixture, or concomitantly with one or more other therapeutic regimens,e.g. either before, simultaneously or after concomitant therapy.

The biological properties of the antagonistic agent may be characterizedex vivo in cell, tissue, and whole organism experiments. As is known inthe art, drugs are often tested in vivo in animals, including but notlimited to mice, rats, rabbits, dogs, cats, pigs, and monkeys, in orderto measure a drug's efficacy for treatment against a disease or diseasemodel, or to measure a drug's pharmacokinetics, pharmacodynamics,toxicity, and other properties. The animals may be referred to asdisease models. Therapeutics are often tested in mice, including but notlimited to nude mice, SCID mice, xenograft mice, and transgenic mice(including knockins and knockouts). Such experimentation may providemeaningful data for determination of the potential of the agent to beused as a therapeutic with the appropriate half-life, effector function,apoptotic activity and IgG inhibitory activity. Any organism, preferablymammals, may be used for testing. For example, because of their geneticsimilarity to humans, primates, monkeys can be suitable therapeuticmodels, and thus may be used to test the efficacy, toxicity,pharmacokinetics, pharmacodynamics, half-life, or other property of theagent. Tests of the substances in humans are ultimately required forapproval as drugs, and these experiments are contemplated herein. Thusthe antagonistic agent of the present invention may be tested in animalmodels or in humans to determine their therapeutic efficacy, toxicity,immunogenicity, pharmacokinetics, and/or other clinical properties.Denosumab is a commercially available product with well-establishedbiological properties, though the anti-pRANKL effect inhibitinginteractions of a cancer cell with thrombocytes turned out to besurprising.

The term “specific” with regard to the RANKL-specific agent as describedherein shall refer to a binding reaction which is determinative of thecognate ligand of interest (RANKL) in a heterogeneous population ofmolecules. Thus, under designated conditions, e.g. immunoassayconditions, the agent that specifically binds to its particular targetdoes not bind in a significant amount to other molecules present in asample.

A specific binding site or a specific agent is typically recognizing thetarget only, and not cross-reactive with other targets. Still, thespecific binding site may specifically bind to one or more epitopes,isoforms or variants of the target, or be cross-reactive to otherrelated target antigens, e.g., homologs or analogs.

The specific binding means that binding is selective in terms of targetidentity, high, medium or low binding affinity or avidity, as selected.Selective binding is usually achieved if the binding constant or bindingdynamics is at least 10 fold different, preferably the difference is atleast 100 fold, and more preferred a least 1000 fold.

The term “surgical intervention” herein also referred to as “surgery”shall refer to a surgical removal, e.g. biopsy, resection or ectomy oftissue comprising all or a part of a tumor, in particular a primarytumor such as a solid tumor and/or one or more metastases.

According to a specific example, the effect of RANKL expression byresting or activated thrombocytes was tested in a lung metastasis mousemodel. In the human system it could be shown that activated thrombocytesexpressed RANKL at a higher level as compared to non-activatedthrombocytes.

According to another example, a RANK-Fc fusion protein was used(Schmiedel et al. 2013, Cancer Res. 73(2):683-94), which is composed ofa human RANK receptor and Fc of human IgG1, and which comprises pointmutations to reduce its affinity to the Fc receptor FcgammaRIIIa, CD16,which are 233P/L234V/L235A/ΔG236/A327G/A330S (nomenclature according toKabat, EU index). The effect of the RANK-Fc fusion protein on metastasisformation in a lung metastasis mouse model was determined. It was shownthat neutralization of RANKL by the RANK-Fc fusion protein was about aseffective as thrombocyte depletion.

Further examples can show that pRANKL is transferred by platelets toRANKL negative tumor cells, thereby transforming the tumor cells toRANKL positive ones. It can also be established that pRANKL inducesprometastatic EMT events in tumor cells, and influences migration oftumor cells through the matrigel. In a mouse knockout modelconditionally knocking out the RANKL expression in megakaryocytes andplatelets, it can be shown that in fact pRANKL induces premetastaticlesions, and pRANKL inhibition would inhibit or reduce metastasisformation.

The foregoing description will be more fully understood with referenceto the following examples. Such examples are, however, merelyrepresentative of methods of practicing one or more embodiments of thepresent invention and should not be read as limiting the scope ofinvention.

EXAMPLES Example 1: RANKL Expression by RANKL Transfected Melanoma Cellsin a Lung Metastasis Mouse Model

Mouse melanoma B16-F10 cells which were transfected with human RANKL orthe parental cell line were used in a mouse model of lung metastasis.Application of transfected, RANKL-positive, cells resulted indrastically enhanced metastatic burden in the lungs of the animals ascompared to the parental cells (control) (FIG. 1). Since it is knownthat human RANKL can stimulate mouse RANK, these data suggest thatincreased metastatic burden in mice which received RANKL-positive tumorcells is due to enhanced RANK signalling into the tumor cells. This isalso in line with studies from Jones et al. (2006) which show enhancedmetastasis of RANK-positive tumor cells upon para- and/or autocrinestimulation. This experiment proves that providing RANKL for RANKstimulation beyond the levels available under normal circumstancesenhances the metastatic potential. The transfection of RANKL thereinmimics the contribution/transfer of pRANKL to the tumor cells.

Example 2: pRANKL Expression on Activated Thrombocytes

Since platelets are known to promote tumor metastasis, analysis ofpotential RANKL surface expression on platelets was performed. While lowlevels of RANKL could be detected on resting platelets, profound RANKLsurface expression was obtained following platelet activation withthrombin (FIG. 2). Thus, activation of platelets, which also occurs uponformation of aggregates with tumor cells, results in rapid upregulationof RANKL expression. The upregulated RANKL is then readily available tointeract and stimulate RANK on tumor cells.

Example 3: RANK-Fc Fusion Protein

The immunoreceptor-Fc fusion protein which contains the extracellularfraction of the human receptor RANK and a human immunoglobulin G (hIgG1)was prepared (FIG. 3). The fusion protein displays markedly reducedaffinity to the Fc receptor (FcγRIIIa, CD16) due to amino acid exchangesin the IgG1 part which prohibits binding of the Fc part to CD16 underphysiological conditions (233P/L234V/L235A/ΔG236/A327G/A330S, Armour etal., 1999, Schmiedel et al., 2013). This construct, in contrast toDenosumab, displays binding to both, human as well as mouse RANKL and istherefore advantageous for usage in murine models of RANKLneutralisation (Bossen et al. 2006, J Biol Chem 281(2):13964-71;Kostenuik et al. 2009, J Bone Miner Res 24(2):182-95).

Example 4: Effects of RANKL Neutralisation in a Lung Metastasis Model

To examine the role of pRANKL which is expressed upon activation (e.g.after coating of circulating tumor cells in the blood stream) in vivo,the melanoma lung metastasis model was applied. Parental melanoma cellswere used to characterize the role of physiologic murine pRANKL in thismodel. The number of metastasis was drastically reduced upon plateletdepletion which is also in line with prior reports. Interestingly,neutralisation of murine RANKL using RANK-Fc-KO fusion proteins resultedin low numbers of metastasis which were comparable to the resultsobtained upon platelet depletion (FIG. 4). These data point to the factthat metastasis is mediated by pRANKL and that a neutralisation thereofcan prevent metastasis formation.

Example 5: Neutralisation of RANKL Prevents Platelet-InducedPrometastatic EMT Signaling in Immortalized MCF10A Cells

As a first step we employed quantitative realtime PCR to exclude thatMCF10A cells, a classical model for EMT analysis, themselves expressRANKL. This served to ascertain that stimulation of tumor-expressed RANKdid not occur in an autocrine/paracrine manner independently ofplatelets. Subsequently MCF10A cells were incubated with human plateletsto mimic platelet-coating of tumor cells in the blood as describedpreviously (Kopp et al. 2009, Cancer Res 69(19):7775-83; Placke et al.2012, Cancer Res 72(2):440-8; and Placke et al. 2012, 189(1):154-60).Cocultures were additionally performed in the presence or absence ofDenosumab to neutralize platelet-derived RANKL. Quantitative real timePCR analysis of the induction of ZEB and NCadherin mRNA demonstratedthat presence of platelets induced expression of the two prometastaticgenes in MCF10A cells. This was largely reduced by blocking RANKL withDenosumab, thereby providing clear evidence that in fact pRANKL mediatesprometastatic effects of platelets on tumor cells upon formation ofaggregates and that neutralization of pRANKL can serve to limit themetastatic potential of tumor cells.

Example 6: Neutralisation of RANKL Prevents Platelet-Induced Migrationof Immortalized MCF10A Cells

Since the migratory potential of malignant cells is key for theirability to form metastasis, we studied whether platelet-derived RANKLalso influenced tumor cell migration. To this end, MCF10A cells wereemployed in a transwell assay system. MCF10A cells were incubated withhuman platelets to mimic platelet-coating of tumor cells in the blood asdescribed above in the presence or absence of isotype control orDenosumab in the upper chamber of a transwell system. After 48 h thenumber of cells that had migrated to the lower chamber along a EGFgradient was determined. We observed that the presence of plateletsclearly enhanced the number of migrated cells, and this was largelyreduced when platelet-derived RANKL was neutralized by the presence ofDenosumab. This demonstrates that pRANKL enhances the migratorypotential of malignant cells and thus their prometastatic phenotype uponformation of aggregates and that neutralization of pRANKL can serve tolimit the metastatic potential of tumor cells (FIG. 7).

Example 7: Lung Metastasis Model Using Platelet-Specific RANKL KnockoutMice

To further examine the role of pRANKL in vivo, the B16-F10 melanoma lungmetastasis model was employed. To specifically assess the role ofplatelet-expressed RANKL, 129-Tnfsf11^(tm1.1Caob)/J mice in which RANKLcontains flox sites (hereinafter referred to as RANKL fl/fl) andC57BL/6-Tg(Pf4-cre)Q3Rsko/J mice which contain a megakariocte/plateletspecific recombinase (hereinafter referred to as Pf4cre) were bred togenerate RANKL fl/fl Pf4 cre/+knockout (ko) mice in which RANKL isspecifically knocked out in megakaryocytes/platelets. For determinationof the effects of platelet-expressed RANKL, B16-F10 melanoma cells(75,000 per mouse) were injected via the tail vein in RANKL fl/fl Pf4cre/+knockout (ko) mice or C57BL/6 control mice (ctrl). The lack ofRANKL in platelets resulted in a substantially reduced number of lungmetastases in the ko as compared to the ctrl group (FIG. 8). These datafurther confirm the specific involvement of pRANKL in metastasisformation in vivo, and support our approach that neutralization ofpRANKL may serve to prevent metastasis.

Example 8: Neutralisation of RANKL Prevents Platelet-InducedPrometastatic EMT Signaling in SK-Mel Melanoma Cells

To confirm and extend the results obtained with immortalized MCF10Acells, we employed the malignant melanoma cell line SK-Mel (ATCC). Flowcytometric analysis excluded that the malignant cells themselvesexpressed RANKL to ascertain that stimulation of tumor-expressed RANKdid not occur in an autocrine/paracrine manner and rather was dependenton platelets (FIG. 9A). In the experiments shown in FIG. 9B, SK-Melcells were incubated with human platelets to mimic platelet-coating oftumor cells in the blood as described before. Cocultures were performedin the presence or absence of Denosumab (5 μg/ml) to neutralizeplatelet-derived RANKL. After 24 h, quantitative real time PCR analysisof the induction of ZEB mRNA demonstrated that, alike with MCF10A cells,presence of platelets induced expression of this prometastatic gene, andthis was largely reduced by blocking platelet-derived RANKL withDenosumab. Similar effects were also observed upon analysis of theexpression levels of Twist and Vimentin, two further genes involved inmetastasis formation/EMT. These analyses confirm and extend our findingsobtained with MCF10A cells described in FIG. 5 and provide furtherevidence that in fact pRANKL mediates prometastatic effects of plateletson tumor cells upon formation of aggregates and that neutralization ofpRANKL can serve to limit the metastatic potential of tumor cells.

1. A method of treating premetastatic lesions in blood of a patent inneed thereof, the method comprising administering a RANKL-specificantagonistic agent recognizing human platelet-expressed receptoractivator of nuclear factor kappa-B ligand (pRANKL).
 2. The method ofclaim 1, wherein the agent is cross-reactive, recognizing pRANKL and atleast one of soluble receptor activator of nuclear factor kappa-B ligand(sRANKL) and membrane-bound activator of nuclear factor kappa-B ligand(mRANKL).
 3. The method of claim, wherein the agent binds to pRANKL,thereby inhibiting pRANKL from activating its receptor on disseminatingcancer cells.
 4. The method of claim 3, wherein the agent is binding topRANKL monomer.
 5. The method of claim 1, wherein the premetastaticlesions are haematogenous.
 6. The method of claim 1, wherein the patientis at risk of or suffering from minimal residual disease and/orrecurrence of metastatic disease.
 7. The method of claim 1, wherein thepatient suffers from a solid tumor selected from the group consisting ofepithelial tumors and mesenchymal tumors, or tumors of endodermal,mesodermal and/or ectodermal origin, or a blood-borne cancer.
 8. Themethod of claim 1, wherein the patient suffers from breast cancer,pancreatic cancer, gastric cancer, esophageal cancer, renal cellcarcinoma, lung carcinoma, colon/rectal/colorectal cancer, melanoma,prostate cancer, head and neck cancer, or leukemia.
 9. The method ofclaim 1, wherein the patient is undergoing surgical intervention toremove at least part of a tumor and/or irradiation, and the agent isadministered for neoadjuvant or adjuvant therapy.
 10. The method ofclaim 1, wherein the agent is selected from the group consisting ofantibodies, antibody fragments, receptor-fusion proteins, peptides,small molecules and aptamers.
 11. The method of claim 1, wherein theagent is a human or humanized antibody, an antigen-binding fragmentthereof, or a RANK-Fc fusion protein.
 12. The method of claim 1, whereinthe agent is administered to the patient in a therapeutically effectiveamount by systemic administration.
 13. The method of claim 1, whereinthe agent is administered to the patient in combination with an adjuvantor neoadjuvant combination therapy.
 14. The method of claim 3, whereinthe agent is binding to pRANKL multimer.
 15. The method of claim 3,wherein the agent is binding to pRANKL, forming a complex with plateletsurface-bound pRANKL or pRANKL cleaved from the platelet surface. 16.The method of claim 1, wherein the premetastatic lesions arehaematogenous as determined by circulating activated platelet-cancercell aggregates.
 17. The method of claim 1, wherein the patient has adetectable level of circulating tumor cells in a blood sample.
 18. Themethod of claim 7, wherein the blood-borne cancer is leukemia.
 19. Themethod of claim 12, wherein the agent is administered to the patient ina therapeutically effective amount by intravenous infusion or bolusinjection.
 20. The method of claim 13, wherein the agent is administeredto the patient in combination with chemotherapy, therapy with kinaseinhibitors and/or immunotherapy.